Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity.

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.

Antibody-directed targeting of retroviral vectors via cell surface antigens.

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1- and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.

Effect of cytokines on HIV-induced depletion of thymocytes in vivo.

BACKGROUND: Cytokines play an important role in the differentiation of thymocytes into mature T cells; consequently, certain cytokines could be useful for immune reconstitution after HIV infection without increasing viral load. OBJECTIVE: To investigate whether cytokines affect immune depletion caused by HIV infection with a CXCR4-tropic strain in SCID-hu mice implanted with human fetal thymus and liver (thy/liv) tissue. METHODS: The thy/liv implants were either mock infected or infected with HIV-1 NL4-3, a CXCR4-tropic molecular clone. Interleukin (IL)-2, IL-4, IL-7, interferon-gamma (IFN-gamma) or diluent was administered to the mice during the second and third week postinfection. Viral load and immunophenotype were determined in thymocytes. RESULTS: Thymocyte subset distributions at 3 weeks postinfection were significantly influenced by treatment with certain cytokines. In particular, IL-2 caused the infected mice to retain a thymocyte profile that was more similar to that in mock-infected mice than that in diluent-treated infected mice, in that the percentages of immature CD4+CD8+ and CD5+CD1+ cells were slightly higher and much less variable than in diluent-treated infected mice. The effect of IFN-gamma treatment was similar to IL-2 but did not reach statistical significance. However, after IFN-gamma treatment, normal percentages of mature CD3+CD69+ cells were maintained whereas this population was relatively increased in diluent-treated infected mice. Although treatment with IL-4 and IL-7 delayed depletion of immature thymocytes, these cytokines increased viral load. CONCLUSIONS: Cytokines such as IL-2 and IFN-gamma maintain immature thymocytes without increasing viral load and may be useful as adjuncts to improve immune reconstitution after HIV infection.

Thymic dendritic cells are primary targets for the oncogenic virus SL3-3.

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70(+) cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70(+) cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70(+) cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70(+) dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.

Regions of human immunodeficiency virus type 1 nef required for function in vivo.

In vivo studies in monkeys and humans have indicated that immunodeficiency viruses with Nef deleted are nonpathogenic in immunocompetent hosts, and this has motivated a search for live attenuated vaccine candidates. However, the mechanisms of action of Nef remain elusive. To define the regions of human immunodeficiency virus type 1 (HIV-1) Nef which mediate in vivo pathogenicity, a series of mutated isogenic viruses were inoculated into human thymic implants in SCID-hu mice. Mutation of several regions, including the myristoylation site at the second glycine and a region encompassing amino acids 41 through 49 of Nef, profoundly affected pathogenicity. Surprisingly, mutations of prolines in either of the two distant PXXP SH3 binding domains did not affect pathogenicity, indicating that these regions are not required for Nef activity in developing T-lineage cells. These data suggest that some functions of Nef described in vitro may not be relevant for in vivo pathogenicity.

Human immunodeficiency virus inhibits multilineage hematopoiesis in vivo.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4(+) lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.

Isolation of mycobacterium-reactive CD1-restricted T cells from patients with human immunodeficiency virus infection.

Because CD1-restricted T cells lack CD4 but produce IFN-gamma in response to nonpeptide mycobacterial antigens, they could play a unique role in immunity to tuberculosis. We studied CD1-restricted T cells in the context of HIV infection by expanding CD4(-) T cell lines from 10 HIV-infected patients. Upon stimulation with Mycobacterium tuberculosis antigen or upon exposure to macrophages infected with M. tuberculosis, these T cell lines proliferated, produced IFN-gamma, and showed cytolytic T cell (CTL) activity against macrophages pulsed with mycobacterial antigen, findings consistent with a protective role against M. tuberculosis. Anti-CD1b antibodies abrogated T cell proliferation, IFN-gamma production, and CTL activity, demonstrating that these T cells are CD1 restricted. IFN-gamma production in response to M. tuberculosis was enhanced by antitransforming growth factor-beta in 8/10 lines, and by IL-15 in 2/10 lines. IFN-gamma production was augmented in a nonantigen-specific manner by IL-12 in 4/8 lines. When live HIV was cocultured with CD1-restricted T cell lines, p24 antigen and proviral DNA were not detected, indicating that the T cells were not infectable with HIV. Vaccination strategies aimed at activation and expansion of M. tuberculosis-reactive CD1-restricted T cells in HIV-infected patients may constitute a novel means to provide protection against tuberculosis, while minimizing the risk of enhancing HIV replication through stimulation of CD4(+) cells.

Preparation and maintenance of SCID-hu mice for HIV research.

The SCID-hu mouse bearing a functional human thymic implant can be easily infected with HIV. Infection results in virus replication and relatively rapid depletion of CD4+ human thymocytes, resulting in a pathologic profile similar to that seen in the thymus of HIV-infected humans. The use of the SCID-hu model for HIV research requires protection of the animals from opportunistic infections and protection of the operators from human pathogens. This discussion describes reliable methods of animal care and surgical procedures to meet these needs.

High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells.

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.

Organization of the V gene segments in mouse T-cell antigen receptor alpha/delta locus.

The mouse T-cell receptor (TCR) alpha/delta locus was mapped using 17 V alpha and 4 V beta subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The alpha/delta locus spans about 1 Mb. The distance between the 3'-most V gene segment (V delta 1) and the delta constant gene (C delta) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual V alpha gene segment subfamilies are dispersed throughout the locus. In contrast, the V delta gene segments V delta 1 to 5 are clustered at the 3' end of the V gene segment cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the alpha/delta locus and on organization features that might influence the expression of specific V gene segments in gamma delta cells.

Contributions to transcriptional activity and to viral leukemogenicity made by sequences within and downstream of the MCF13 murine leukemia virus enhancer.

We have identified nucleotide sequences that regulate transcription in both a cell-type-specific and general manner in the long terminal repeat of the MCF13 murine leukemia virus. Besides the enhancer element, we have observed that the region between the enhancer and promoter (DEN) has a profound effect on transcription in different cell types. This effect, however, was dependent on the copy number of enhancer repeats and was detectable in the presence of a single repeat. When two enhancer repeats were present, the effect of DEN on transcription was abrogated except in T cells. DEN also makes a significant contribution to the leukemogenic property of the MCF13 retrovirus. Its deletion from the MCF13 virus dramatically reduced the incidence of thymic lymphoma and increased the latency of disease in comparison with the wild-type virus. This effect was most marked when one rather than two enhancer repeats was present in the mutant viruses. We also observed that the removal of one repeat alone remarkably reduced leukemogenicity by the MCF13 virus. A newly identified protein-binding site (MLPal) located within DEN affects transcription only in T cells, and its deletion attenuates the ability of an MCF13 virus with a single enhancer repeat to induce thymic lymphoma. This observation suggests that the MLPal protein-binding site contributes to the effect of the DEN region on T-cell-specific transcription and viral leukemogenicity. This study identifies the importance of nonenhancer sequences in the long terminal repeat for the oncogenesis of the MCF13 retrovirus.

Observations on lymphomagenesis and lymphoma in AKR mice. A description of prelymphoma changes in the thymus and phenotypic diversity of lymphomas induced by SL3-3 virus.

These studies were designed to look for a correlation of intrathymic survival of virus-infected thymocytes with lymphomagenesis. Cells from the normal-appearing prelymphoma thymus of SL3-3 virus-treated AKR mice were studied. Also, phenotypic properties of the malignant cells from the virus-induced lymphomas are described. In this model system, 100% of mice inoculated with virus at three days of age develop thymic lymphoma between 60 and 90 days of age. The experiments show that cells with malignant potential do not appear in the thymus until 36 days after virus inoculation. These cells are initially thymus-dependent (TD) in that they produce lymphoma of donor-type in recipients after intrathymic inoculation with long latency. They do not produce lymphoma after subcutaneous inoculation in syngeneic hosts. At 39 days after virus inoculation, the first thymus independent (TI) lymphoma cells appear. These cells, like the cells isolated from thymi with overt tumors, produce lymphoma of donor-type after a short latency when inoculated by the intrathymic or subcutaneous route. Thymocytes from normal-appearing thymi of mice at 42 days after virus inoculation, which could be expected to include TD, TI or no lymphoma cells, were evaluated for their ability to survive in a recipient thymus for three weeks after intrathymic inoculation. They were compared to thymocytes from age-matched control mice. Thymi receiving the virus-infected thymocytes showed 15% to 80% donor cells at three weeks. The highest numbers of donor cells were from thymi which were shown to contain TI lymphoma cells. However, cells from thymi with TD and no lymphoma cells could also be detected in significant numbers at three weeks after intrathymic inoculation. Less than 2% of donor-type thymocytes could be found after inoculation of thymocytes from normal control AKR mice. These data provide evidence that virus infection of thymocytes, even before the appearance of cells with lymphomagenic potential, endows them with a capacity for prolonged intrathymic survival. This appears to be a necessary step for tumor progression in this model. A remarkable phenotypic diversity of the virus-induced lymphomas was shown. The effect of various growth environments, intrathymic, subcutaneous, and in vitro on lymphoma cell phenotypic expression revealed individual differences in each tumor and in each environment.

Mechanisms of thymic lymphomagenesis by the retrovirus SL3-3.

These studies report changes occurring in the thymus of AKR and NFS/N mice after infection with the lymphomagenic retrovirus SL3-3. In virus-infected AKR fetal thymus, the programmed cell death caused by treatment with antibody to CD3 was remarkably diminished. A method of establishing thymic stromal cultures from mice of 1 to 3 wk of age is described. Using this method, it was found that SL3-3 virus infection by neonatal inoculation allowed establishment of thymic stromal cultures from organs removed from AKR mice of 30 to 50 days of age and from lymphomas, whereas thymic stromal cultures could not be established from control mice after 30 days of age. Using NFS/N mice which have no endogenous virus, it was shown that infection of thymic stroma precedes infection of thymocytes and that thymocytes are permissive for infection with SL3-3 virus but not for the nononcogenic retrovirus, Akv, yet Akv virus replicates efficiently in thymic stroma. SL3-3 virus integrates randomly in each lymphoma induced by this virus. The lymphomas are clonal or oligoclonal. Pim-1 and c-myc genes commonly rearranged in other virus-induced thymic lymphoma showed rearrangement in only a few lymphomas. A theory is proposed, based on the work presented here and in recent studies, which states that SL3-3 virus infection of thymic stroma allows infection of thymocyte progenitors entering from the bone marrow. These cells are then altered so that their maturation is delayed and their intrathymic survival is prolonged. This permits virus integration and reintegration that results in the genetic changes which transform the cell.

Progression to development of lymphoma in the thymus of AKR mice treated neonatally with SL 3-3 virus.

All AKR mice develop thymic lymphoma between 60 and 90 days of age after neonatal treatment with the oncogenic retrovirus SL 3-3. At 40-50 days of age, in the normal-sized thymus of virus-treated mice, cells appear that produce lymphoma when inoculated intrathymically but not when inoculated s.c. These cells are designated as thymus-dependent (TD) lymphoma cells. TD cells progress to cells that form tumors after both intrathymic and s.c. inoculation; these are designated as thymus-independent (TI) lymphoma cells. In this report, we show that the TD and TI cells can be distinguished as two distinct cell populations. Experiments show that the TD cells reside within the immature CD4- CD8- thymocyte population of the virus-treated mice. In addition, we also show that CD4- CD8- thymocytes from SL 3-3 virus-treated mice do not mature in fetal thymic stromal rudiments. Using three-color flow cytometry to trace maturation of CD4- CD8- thymocytes after intrathymic inoculation into irradiated syngeneic hosts, disregulated thymocyte maturation of this population from virus-treated mice is demonstrated. Thus, altered maturation of and the appearance of TD lymphoma cells in, the most immature population of thymocytes appears to be a first step in a multistep process of thymic lymphomagenesis caused by SL 3-3 virus.

Development of lymphoma in the thymus of AKR mice treated with the lymphomagenic virus SL 3-3.

A chronological study of the individual thymic lobes of young AKR mice after neonatal inoculation of the oncogenic AKR retrovirus SL 3-3 was performed. 100% of mice treated in this manner develop lymphoma between 60 and 100 days of age. A search for early lymphoma cells in individual thymi was carried out by inoculating the thymocytes subcutaneously in syngeneic and intrathymically in syngeneic and semisyngeneic recipients. Tumor progression was observed in animals between 48 and 60 days of age. These animals have: (a) normal weight lobes, in which no lymphoma cells could be detected, (b) thymus-dependent lymphoma cells, in one or both normal weight lobes; (c) thymus-independent lymphoma cells, found in lobes of normal weight as well as in thymi enlarged by lymphoma cells. Thymocyte characteristics of virus-treated animals of 21 to 63 days of age were compared with those of age-matched controls. Beginning at 28 days a concordant, progressive with time, increase of thymocyte surface staining for the viral envelope glycoprotein gp70 was seen in all lobes from virus-treated animals. Evaluation of cell surface markers by two-color fluorescence with antibodies to CD4 and CD8 showed that after 50 days of age, thymic lobes with and without lymphomas had nonspecific, but marked, alterations of the typical thymocyte surface marker pattern. No characteristic CD4, CD8 surface phenotype was found in primary lymphomas. Using probes for the T-cell receptor J beta 2 gene segments and the Akv ecotropic virus gp70 envelope genes, oligoclonality in J beta 2 rearrangements and clonality using the Akv env genes was demonstrated in thymi with the thymus-dependent phenotype. In lymphomas T-cell receptor beta gene probes showed either oligoclonality or clonality. Clonal virus integrations were found in these lymphomas. These experiments suggest the following series of events in virus-accelerated AKR lymphomagenesis. First, lymphoma cells arise which are initially thymus-dependent and can appear in one or simultaneously in both thymic lobes. These progress to become thymus-independent, fully autonomous, tumor cells. Thymocytes close to or at the time of the initial transformation event show a marked disorder of differentiation defined by the alterations in the CD4, CD8 surface phenotype distribution.

Metal-dependent binding of a factor in vivo to the metal-responsive elements of the metallothionein 1 gene promoter.

Using the technique of genomic footprinting, we demonstrate cadmium-inducible protection from dimethyl sulfate (DMS) modification of guanine residues in vivo in five metal-responsive elements (MREs) in the promoter of the rat metallothionein 1 (MT-1) gene. We also identify a site of extreme DMS hyperreactivity which, like the MRE protection, occurs only after metal ion induction. With this hyperreactive site as an indicator, we can measure the kinetics of induction and deinduction. Changes in the intracellular metal ion concentrations are reflected in alterations in the reactivity with DMS of guanine residues in the MT-1 gene promoter. Lastly, for both control and metal-induced cells, we observe DMS protection and enhancement of a binding site (located 5' of the distal MRE) which is a consensus sequence for the Sp1 transcription factor. Transfection experiments with deletion mutations of a fusion gene construct indicate both that a sequence region which includes this GC box regulates the basal level of expression of the MT-1 gene and that increasing the number of MREs in the promoter increases the induced level of transcription. Our genomic footprinting and transfection data together suggest that (i) a transcription factor, possibly Sp1, plays an important role in regulating the basal level of expression of the MT-1 gene and (ii) metal induction involves the metal-dependent binding to a sequence-specific binding factor which responds to changes in intracellular metal ion levels.

Rat metallothionein multigene family.

Southern blot analysis of rat genomic DNA reveals the presence of numerous sequences homologous to the rat MT-1 gene. We have isolated and characterized by sequence analysis the rat MT-1 gene and three related processed pseudogenes. We discuss mechanisms by which these pseudogenes were formed. The rat MT-1 and MT-2 structural genes are shown to be separated by 4 kb of intergenic DNA. By sequence analysis of the MT-2 structural gene, we compare features of the MT gene sequences which regulate promoter activity, and which specify transcription termination and polyadenylation. From an examination of homologous sequences in the MT promoters, we suggest that there may be both regulatory features in common with the MT gene isotypes, and regulatory mechanisms which may allow for differential expression of these genes. We discuss possible applications of recombinant DNA technology to investigating both the expression of MT genes and the functioning of MT proteins, in a tissue specific manner and during development.

A video-computer for the chronotropic and inotropic measurements of the beating of cultured heart cells.

A video-computer system has been developed to measure the chronotropy and inotropy of cultured heart cells. The motion of the cell is followed by recording of the changes of light intensity from the edge of a beating cell. With this system the velocity of contraction and relaxation, the time to peak contraction, relaxation time and the displacement of the cell in culture can be measured for the first time. Also, the rate of beating can be measured beyond the limits set by visual counting. With the application of this technique to the cultured heart cell system we have found that norepinephrine increases the velocity of relaxation thus reducing the ratio of contraction velocity to relaxation velocity, and reduces the twitch time. Increased external calcium, on the other hand, has little effect on either the velocity of contraction or relaxation but, like norepinephrine, decreases the twitch time.

Effect of parathyroid hormone on osmotic fragility of human erythrocytes.

The survival of erythrocytes (RBC) is shortened in uremia, and it has been shown that calcium influx into RBC evoked crenation and increased their rigidity. The high blood levels of parathyroid hormone (PTH) may augment entry of calcium into RBC and hence affect their integrity. We examined the effect of PTH on osmotic fragility of human RBC and investigated the mechanisms through which PTH interacts with RBC. Both the amino-terminal (1-34) PTH and the intact (1-84) PTH, but not the carboxy-terminal (53-84) PTH, produced significant increases in osmotic fragility. This effect was abolished by prior inactivation of the hormone. There was a dose-response relationship between both moieties of PTH and the increase in osmotic fragility. This action of PTH required calcium, was mimicked by calcium ionophore, and was partially blocked by verapamil. PTH caused significant influx of (45)Ca into RBC, which was not associated with potassium leak. The hormone did not affect water content of RBC. Scanning electron microscopy revealed that the incubation of RBC with PTH was associated with the appearance of membrane filamentous extensions, which anchor RBC together. Inhibition of glycolytic activity of RBC with NaF or inhibition of Na-K-activated ATPase with ouabain did not abolish the effect of PTH on osmotic fragility. PTH did not stimulate RBC Na-K-activated ATPase or Mg-dependent ATPase but caused marked and significant stimulation of Ca-activated ATPase. The basal activity of the RBC adenylate cyclase was low and PTH produced only a modest stimulation of this enzyme. Both cyclic AMP and dibutyryl cyclic AMP had no effect on osmotic fragility. THE DATA INDICATE THAT: (a) the RBC is a target organ for PTH, (b) the hormone increases osmotic fragility of RBC, and (c) this effect of PTH is due to enhanced calcium entry into RBC. We suggest that the increased calcium influx may affect the spectrin-actin of the cytoskeletal network of the RBC and may alter the stability and integrity of the cell membrane. This action of PTH on the RBC could be, at least in part, responsible for the shortened survival of RBC in uremia, and assign a new role for PTH in the pathogenesis of the anemia of uremia.